The protozoan Trypanosoma cruzi was discovered as the etiologic agent of Chagas' disease over 100 years ago, yet many of the basic aspects of the parasite-host interaction remain unknown. As an obligate intracellular parasite that lives in the cytoplasm of its mammalian host cell, T. cruzi intersects a variety of host cellular networks to promote its replication and long-term survival in the host. The overarching goal of our research is to delineate host factors that are critical for supporting intracellular T. cruzi infection, wit a view to identifying avenues to pharmacologically uncouple this important human pathogen from its host. The focus of this proposed study is the host cytoskeleton. Preliminary observations indicate that cytosolically-localized parasites track spatially with the major cytoskeletal network in the host cell suggestive of physical interactions between T. cruzi and components of the host cytoskeleton. To characterize this interaction, live imaging will be employed to chart the dynamics and spatial organization of T. cruzi amastigotes in mammalian cells that are undergoing dramatic cytoskeletal reorganization following trypsinization and replating. Adhesive fibronectin micropatterns will then be exploited to limit morphological heterogeneity of the infected host cell population and standardize intracellular patterning of T. cruzi. Using these standardized conditions we will conduct a small-scale RNA interference (RNAi) screen to identify host factors that mediate parasite-host cytoskeletal interactions. In line with our prediction that tethering of T. cruzi amastigotes to the host cytoskeleton impacts the replicative capacity of the parasite, functional studies will focus on host genes previously identified in our genome-scale RNAi screen to have demonstrated impact on host metabolism and parasite infectivity, including a subset of cytoskeletal genes. This study opens a new direction in the T. cruzi field that explores the intersection of host cytoskeleton, cell shape, and metabolism and T. cruzi amastigote proliferation.